This invention relates to novel nucleotide triphosphates (NTPs); methods for synthesizing nucleotide triphosphates; and methods for incorporation of novel nucleotide triphosphates into oligonucleotides. The invention further relates to incorporation of these nucleotide triphosphates into nucleic acid molecules using polymerases under several novel reaction conditions.
The following is a brief description of nucleotide triphosphates. This summary is not meant to be complete, but is provided only to assist understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
The synthesis of nucleotide triphosphates and their incorporation into nucleic acids using polymerase enzymes has greatly assisted in the advancement of nucleic acid research. The polymerase enzyme utilizes nucleotide triphosphates as precursor molecules to assemble oligonucleotides. Each nucleotide is attached by a phosphodiester bond formed through nucleophilic attack by the 3xe2x80x2 hydroxyl group of the oligonucleotide""s last nucleotide onto the 5xe2x80x2 triphosphate of the next nucleotide. Nucleotides are incorporated one at a time into the oligonucleotide in a 5xe2x80x2 to 3xe2x80x2 direction. This process allows RNA to be produced and amplified from virtually any DNA or RNA templates.
Most natural polymerase enzymes incorporate standard nucleotide triphosphates into nucleic acid. For example, a DNA polymerase incorporates dATP, dTTP, dCTP, and dGTP into DNA and an RNA polymerase generally incorporates ATP, CTP, UTP, and GTP into RNA. There are however, certain polymerases that are capable of incorporating non-standard nucleotide triphosphates into nucleic acids (Joyce, 1997, PNAS 94, 1619-1622, Huang et al., Biochemistry 36, 8231-8242).
Before nucleosides can be incorporated into RNA transcripts using polymerase enzymes they must first be converted into nucleotide triphosphates which can be recognized by these enzymes. Phosphorylation of unblocked nucleosides by treatment with POCl3 and trialkyl phosphates was shown to yield nucleoside 5xe2x80x2-phosphorodichloridates (Yoshikawa et al., 1969, Bull. Chem. Soc.(Japan) 42, 3505). Adenosine or 2xe2x80x2-deoxyadenosine 5xe2x80x2-triphosphate was synthesized by adding an additional step consisting of treatment with excess tri-n-butylammonium pyrophosphate in DMF followed by hydrolysis (Ludwig, 1981, Acta Biochim. et Biophys. Acad. Sci. Hung. 16, 131-133).
Non-standard nucleotide triphosphates are not readily incorporated into RNA transcripts by traditional RNA polymerases. Mutations have been introduced into RNA polymerase to facilitate incorporation of deoxyribonucleotides into RNA (Sousa and Padilla, 1995, EMBO J. 14,4609-4621, Bonner et al., 1992, EMBO J. 11, 3767-3775, Bonner et al., 1994, J Biol. Chem. 42, 25120-25128, Aurup et al., 1992, Biochemistry 31, 9636-9641).
McGee et al., International PCT Publication No. WO 95/35102, describes the incorporation of 2xe2x80x2-NH2-NTP""s, 2xe2x80x2-F-NTP""s, and 2xe2x80x2-deoxy-2xe2x80x2-benzyloxyamino UTP into RNA using bacteriophage T7 polymerase.
Wieczorek et al., 1994, Bioorganic and Medicinal Chemistry Letters 4, 987-994, describes the incorporation of 7-deaza-adenosine triphosphate into an RNA transcript using bacteriophage T7 RNA polymerase.
Lin et al., 1994, Nucleic Acids Research 22, 5229-5234, reports the incorporation of 2xe2x80x2-NH2-CTP and 2xe2x80x2-NH2-UTP into RNA using bacteriophage T7 RNA polymerase and polyethylene glycol containing buffer. The article describes the use of the polymerase synthesized RNA for in vitro selection of aptamers to human neutrophil elastase (HNE).
This invention relates to novel nucleotide triphosphate (NTP) molecules, and their incorporation into nucleic acid molecules, including nucleic acid catalysts. The NTPs of the instant invention are distinct from other NTPs known in the art. The invention further relates to incorporation of these nucleotide triphosphates into oligonucleotides using an RNA polymerase; the invention further relates to novel transcription conditions for the incorporation of modified (non-standard) and unmodified NTP""s into nucleic acid molecules. Further, the invention relates to methods for synthesis of novel NTP""s
In a first aspect, the invention features NTP""s having the formula triphosphate-OR, for example the following formula I: 
where R is any nucleoside; specifically the nucleosides 2xe2x80x2-O-methyl-2,6-diaminopurine riboside; 2xe2x80x2-deoxy-2xe2x80x2-amino-2,6-diaminopurine riboside; 2xe2x80x2-(N-alanyl)amino-2xe2x80x2-deoxy-uridine; 2xe2x80x2-(N-phenylalanyl)amino-2xe2x80x2-deoxy-uridine; 2xe2x80x2-deoxy-2xe2x80x2-(N-xcex2-alanyl)amino; 2xe2x80x2-deoxy-2xe2x80x2-(lysiyl)amino uridine; 2xe2x80x2-C-allyl uridine; 2xe2x80x2-O-amino-uridine; 2xe2x80x2-O-methylthiomethyl adenosine; 2xe2x80x2-O-methylthiomethyl cytidine; 2xe2x80x2-O-methylthiomethyl guanosine; 2xe2x80x2-O-methylthiomethyl-uridine; 2xe2x80x2-deoxy-2xe2x80x2-(N-histidyl)amino uridine; 2xe2x80x2-deoxy-2xe2x80x2-amino-5-methyl cytidine; 2xe2x80x2-(N-xcex2-carboxamidine-xcex2-alanyl)amino-2xe2x80x2-deoxy-uridine; 2xe2x80x2-deoxy-2xe2x80x2-(N-xcex2-alanyl)-guanosine; 2xe2x80x2-O-amino-adenosine; 2xe2x80x2-(N-lysyl)amino-2xe2x80x2-deoxy-cytidine; 2xe2x80x2-Deoxy-2xe2x80x2-(L-histidine)amino Cytidine; 5-Imidazoleacetic acid 2xe2x80x2-deoxy uridine, 5-[3-(N-4-imidazoleacetyl)aminopropynyl]-2xe2x80x2-O-methyl uridine, 5-(3-aminopropynyl)-2xe2x80x2-O-methyl uridine, 5-(3-aminopropyl)-2xe2x80x2-O-methyl uridine, 5-[3-(N-4-imidazoleacetyl)aminopropyl]-2xe2x80x2-O-methyl uridine, 5-(3-aminopropyl)-2xe2x80x2-deoxy-2-fluoro uridine, 2xe2x80x2-Deoxy-2xe2x80x2-(xcex2-alanyl-L-histidyl)amino uridine, 2xe2x80x2-deoxy-2xe2x80x2-xcex2-alaninamido-uridine, 3-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-ribofuranosyl)piperazino[2,3-D]pyrimidine-2-one, 5-[3-(N-4-imidazoleacetyl)aminopropyl]-2xe2x80x2-deoxy-2xe2x80x2-fluoro uridine, 5-[3-(N-4-imidazoleacetyl)aminopropynyl]-2xe2x80x2-deoxy-2xe2x80x2-fluoro uridine, 5-E-(2-carboxyvinyl-2xe2x80x2-deoxy-2xe2x80x2-fluoro uridine, 5-[3-(N-4-aspartyl)aminopropynyl-2xe2x80x2-fluoro uridine, 5-(3-aminopropyl)-2xe2x80x2-deoxy-2-fluoro cytidine, and 5-[3-(N-4-succynyl)aminopropyl-2xe2x80x2-deoxy-2-fluoro cytidine.
In a second aspect, the invention features inorganic and organic salts of the nucleoside triphosphates of the instant invention.
In a third aspect, the invention features a process for the synthesis of pyrimidine nucleotide triphosphate (such as UTP, 2xe2x80x2-O-MTM-UTP, dUTP and the like) including the steps of monophosphorylation where the pyrimidine nucleoside is contacted with a mixture having a phosphorylating agent (such as phosphorus oxychloride, phospho-tris-trizolides, phospho-tris-triimidazolides and the like), trialkyl phosphate (such as triethylphosphate or trimethylphosphate or the like) and a hindered base (such as dimethylaminopyridine, DMAP and the like) under conditions suitable for the formation of pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine monophosphate is contacted with a pyrophosphorylating reagent (such as tributylammonium pyrophosphate) under conditions suitable for the formation of pyrimidine triphosphates.
The term xe2x80x9cnucleotidexe2x80x9d as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1xe2x80x2 position of a sugar moiety. Nucleotides generally include a base, a sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; all hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art, e.g., as recently summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine) and others (Burgin et al., 1996, Biochemistry, 35, 14090). By xe2x80x9cmodified basesxe2x80x9d in this aspect is meant nucleotide bases other than adenine, guanine, cytosine thymine and uracil at 1xe2x80x2 position or their equivalents; such bases may be used within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of such a molecule. Such modified nucleotides include dideoxynucleotides which have pharmaceutical utility well known in the art, as well as utility in basic molecular biology methods such as sequencing.
By xe2x80x9cribonucleotidexe2x80x9d is meant a nucleotide with a hydroxyl group at the 2xe2x80x2 position of a xcex2-D-ribo-furanose moiety.
By xe2x80x9cunmodified nucleosidexe2x80x9d or xe2x80x9cunmodified nucleotidexe2x80x9d is meant one of the bases adenine, cytosine, guanine, uracil joined to the 1xe2x80x2 carbon of xcex2-D-ribo-furanose with substitutions on either moiety.
By xe2x80x9cmodified nucleosidexe2x80x9d or xe2x80x9cmodified nucleotidexe2x80x9d is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
By xe2x80x9cpyrimidinesxe2x80x9d is meant nucleotides comprising modified or unmodified derivatives of a six membered pyrimidine ring. An example of a pyrimidine is modified or unmodified uridine.
By xe2x80x9cnucleotide triphosphatexe2x80x9d or xe2x80x9cNTPxe2x80x9d is meant a nucleoside bound to three inorganic phosphate groups at the 5xe2x80x2 hydroxyl group of the modified or unmodified ribose or deoxyribose sugar where the 1xe2x80x2 position of the sugar may comprise a nucleic acid base or hydrogen. The triphosphate portion may be modified to include chemical moieties which do not destroy the functionality of the group (i.e., allow incorporation into an RNA molecule).
In another preferred embodiment, nucleotide triphosphates (NTPs) of the instant invention are incorporated into an oligonucleotide using an RNA polymerase enzyme. RNA polymerases include but are not limited to mutated and wild type versions of bacteriophage T7, SP6, or T3 RNA polymerases. Applicant has also found that the NTPs of the present invention can be incorporated into oligonucleotides using certain DNA polymerases, such as Taq polymerase.
In yet another preferred embodiment, the invention features a process for incorporating modified NTP""s into an oligonucleotide including the step of incubating a mixture having a DNA template, RNA polymerase, NTP, and an enhancer of modified NTP incorporation under conditions suitable for the incorporation of the modified NTP into the oligonucleotide.
By xe2x80x9cenhancer of modified NTP incorporationxe2x80x9d is meant a reagent which facilitates the incorporation of modified nucleotides into a nucleic acid transcript by an RNA polymerase. Such reagents include but are not limited to methanol; LiCl; polyethylene glycol (PEG); diethyl ether; propanol; methyl amine; ethanol and the like.
In another preferred embodiment, the modified nucleotide triphosphates can be incorporated by transcription into a nucleic acid molecules including enzymatic nucleic acid, antisense, 2-5A antisense chimera, oligonucleotides, triplex forming oligonucleotide (TFO), aptamers and the like (Stull et al., 1995 Pharmaceutical Res. 12, 465).
By xe2x80x9cantisensexe2x80x9d it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004; Agrawal et al., U.S. Pat. No. 5,591,721; Agrawal, U.S. Pat. No. 5,652,356). Typically, antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop. Thus, the antisense molecule may be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule may be complementary to a target sequence or both.
By xe2x80x9c2-5A antisense chimeraxe2x80x9d it is meant, an antisense oligonucleotide containing a 5xe2x80x2 phosphorylated 2xe2x80x2-5xe2x80x2-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
By xe2x80x9ctriplex forming oligonucleotides (TFO)xe2x80x9d it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504).
By xe2x80x9coligonucleotidexe2x80x9d as used herein is meant a molecule having two or more nucleotides. The polynucleotide can be single, double or multiple stranded and may have modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
By xe2x80x9cnucleic acid catalystxe2x80x9d is meant a nucleic acid molecule capable of catalyzing (altering the velocity and/or rate of) a variety of reactions including the ability to repeatedly cleave other separate nucleic acid molecules (endonuclease activity) in a nucleotide base sequence-specific manner. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme, finderon or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).
By xe2x80x9cenzymatic portionxe2x80x9d or xe2x80x9ccatalytic domainxe2x80x9d is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate.
By xe2x80x9csubstrate binding armxe2x80x9d or xe2x80x9csubstrate binding domainxe2x80x9d is meant that portion/region of an enzymatic nucleic acid molecule which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. That is, these arms contain sequences within a enzymatic nucleic acid molecule which are intended to bring enzymatic nucleic acid molecule and target together through complementary base-pairing interactions. The enzymatic nucleic acid molecule of the invention may have binding arms that are contiguous or non-contiguous and may be varying lengths. The length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
By xe2x80x9cnucleic acid moleculexe2x80x9d as used herein is meant a molecule having nucleotides. The nucleic acid can be single, double or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. An example of a nucleic acid molecule according to the invention is a gene which encodes for a macromolecule such as a protein.
In preferred embodiments of the present invention, a nucleic acid molecule, e.g., an antisense molecule, a triplex DNA, or an enzymatic nucleic acid molecule, is 13 to 100 nucleotides in length, e.g., in specific embodiments 35, 36, 37, or 38 nucleotides in length (e.g., for particular ribozymes). In particular embodiments, the nucleic acid molecule is 15-100, 17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100, 32-100, 35-100, 40-100, 50-100, 60-100, 70-100, or 80-100 nucleotides in length. Instead of 100 nucleotides being the upper limit on the length ranges specified above, the upper limit of the length range can be, for example, 30, 40, 50, 60, 70, or 80 nucleotides. Thus, for any of the length ranges, the length range for particular embodiments has lower limit as specified, with an upper limit as specified which is greater than the lower limit. For example, in a particular embodiment, the length range can be 35-50 nucleotides in length. All such ranges are expressly included. Also in particular embodiments, a nucleic acid molecule can have a length which is any of the lengths specified above, for example, 21 nucleotides in length.
By xe2x80x9ccomplementarityxe2x80x9d is meant that a nucleic acid can form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well-known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785. A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). xe2x80x9cPerfectly complementaryxe2x80x9d means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
In yet another preferred embodiment, the modified nucleotide triphosphates of the instant invention can be used for combinatorial chemistry or in vitro selection of nucleic acid molecules with novel function. Modified oligonucleotides can be enzymatically synthesized to generate libraries for screening.
In another preferred embodiment, the invention features nucleic acid based techniques (e.g., enzymatic nucleic acid molecules), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) isolated using the methods described in this invention and methods for their use to diagnose, down regulate or inhibit gene expression.
In yet another perferred embodiment, the invention features enzymatic nucleic acid molecules targeted against HER2 RNA, specifically including ribozymes in the class II (zinzyme) motif.
By xe2x80x9cinhibitxe2x80x9d it is meant that the activity of target genes or level of mRNAs or equivalent RNAs encoding target genes is reduced below that observed in the absence of the nucleic acid molecules of the instant invention (e.g., enzymatic nucleic acid molecules), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups). In one embodiment, inhibition with enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically attenuated nucleic acid molecule that is able to bind to the same site on the mRNA, but is unable to cleave that RNA. In another embodiment, inhibition with nucleic acid molecules, including enzymatic nucleic acid and antisense molecules, is preferably greater than that observed in the presence of for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition of target genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absence.
In yet another preferred embodiment, the invention features a process for incorporating a plurality of compounds of formula I.
In yet another embodiment, the invention features a nucleic acid molecule with catalytic activity having formula II: 
In the formula shown above X, Y, and Z represent independently a nucleotide or a non-nucleotide linker, which may be same or different; . indicates hydrogen bond formation between two adjacent nucleotides which may or may not be present; Yxe2x80x2 is a nucleotide complementary to Y; Zxe2x80x2 is a nucleotide complementary to Z; l is an integer greater than or equal to 3 and preferably less than 20, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; m is an integer greater than 1 and preferably less than 10, more specifically 2, 3, 4, 5, 6, or 7; n is an integer greater than 1 and preferably less than 10, more specifically 3, 4, 5, 6, or 7; o is an integer greater than or equal to 3 and preferably less than 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; l and o may be the same length (l=o) or different lengths (lxe2x89xa0o); each X(l) and X(o) are oligonucleotides which are of sufficient length to stably interact independently with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); W is a linker of xe2x89xa72 nucleotides in length or may be a non-nucleotide linker; A, U, C, and G represent the nucleotides; G is a nucleotide, preferably 2xe2x80x2-O-methyl or ribo; A is a nucleotide, preferably 2xe2x80x2-O-methyl or ribo; U is a nucleotide, preferably 2xe2x80x2-amino (e.g., 2xe2x80x2-NH2 or 2xe2x80x2-Oxe2x80x94NH2), 2xe2x80x2-O-methyl or ribo; C represents a nucleotide, preferably 2xe2x80x2-amino (e.g., 2xe2x80x2-NH2 or 2xe2x80x2-Oxe2x80x94NH2), and _ represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate or others known in the art).
In yet another embodiment, the invention features a nucleic acid molecule with catalytic activity having formula III (SEQ ID NO: 1519): 
In the formula shown above X, Y, and Z represent independently a nucleotide or a non-nucleotide linker, which may be same or different; . indicates hydrogen bond formation between two adjacent nucleotides which may or may not be present; Zxe2x80x2 is a nucleotide complementary to Z; l is an integer greater than or equal to 3 and preferably less than 20, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; n is an integer greater than 1 and preferably less than 10, more specifically 3, 4, 5, 6, or 7; o is an integer greater than or equal to 3 and preferably less than 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; l and o may be the same length (l=o) or different lengths (lxe2x89xa0o); each X(l) and X(o) are oligonucleotides which are of sufficient length to stably interact independently with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); X(o) preferably has a G at the 3xe2x80x2-end, X(l) preferably has a G at the 5xe2x80x2-end; W is a linker of xe2x89xa72 nucleotides in length or may be a non-nucleotide linker; Y is a linker of xe2x89xa71 nucleotides in length, preferably G, 5xe2x80x2-CA-3xe2x80x2, or 5xe2x80x2-CAA-3xe2x80x2, or may be a non-nucleotide linker; A, U, C, and G represent the nucleotides; G is a nucleotide, preferably 2xe2x80x2-O-methyl, 2xe2x80x2-deozy-2xe2x80x2-fluoro, or 2xe2x80x2-OH; A is a nucleotide, preferably 2xe2x80x2-O-methyl, 2xe2x80x2-deozy-2xe2x80x2-fluoro, or 2xe2x80x2-OH; U is a nucleotide, preferably 2xe2x80x2-O-methyl, 2xe2x80x2-deozy-2xe2x80x2-fluoro, or 2xe2x80x2-OH; C represents a nucleotide, preferably 2xe2x80x2-amino (e.g., 2xe2x80x2-NH2 or 2xe2x80x2-Oxe2x80x94NH2, and _ represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate or others known in the art).
The enzymatic nucleic acid molecules of Formula II and Formula III may independently comprise a cap structure which may independently be present or absent.
By xe2x80x9csufficient lengthxe2x80x9d is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, for binding arms of enzymatic nucleic acid xe2x80x9csufficient lengthxe2x80x9d means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. Preferably, the binding arms are not so long as to prevent useful turnover.
By xe2x80x9cstably interactxe2x80x9d is meant, interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions).
By xe2x80x9cchimeric nucleic acid moleculexe2x80x9d or xe2x80x9cchimeric oligonucleotidexe2x80x9d is meant that, the molecule may be comprised of both modified or unmodified DNA or RNA.
By xe2x80x9ccap structurexe2x80x9d is meant chemical modifications, which have been incorporated at a terminus of the oligonucleotide. These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5xe2x80x2-terminus (5xe2x80x2-cap) or at the 3xe2x80x2-terminus (3xe2x80x2-cap) or may be present on both termini. In non-limiting examples the 5xe2x80x2-cap is selected from the group comprising inverted abasic residue (moiety), 4xe2x80x2,5xe2x80x2-methylene nucleotide, 1-(beta-D-erythrofuranosyl)nucleotide, 4xe2x80x2-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides, modified base nucleotide, phosphorodithioate linkage, threo-pentofuranosyl nucleotide, acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3xe2x80x2-3xe2x80x2-inverted nucleotide moiety, 3xe2x80x2-3xe2x80x2-inverted abasic moiety; 3xe2x80x2-2xe2x80x2-inverted nucleotide moiety; 3xe2x80x2-2xe2x80x2-inverted abasic moiety; 1,4-butanediol phosphate, 3xe2x80x2-phosphoramidate, hexylphosphate, aminohexyl phosphate; 3xe2x80x2-phosphate, 3xe2x80x2-phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety (for more details see Beigelman et al., International PCT publication No. WO 97/26270, incorporated by reference herein). In yet another preferred embodiment the 3xe2x80x2-cap is selected from a group comprising, 4xe2x80x2,5xe2x80x2-methylene nucleotide, 1-(beta-D-erythrofuranosyl)nucleotide, 4xe2x80x2-thio nucleotide, carbocyclic nucleotide, 5xe2x80x2-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, phosphorodithioate, threo-pentofuranosyl nucleotide, acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide, 3,4-dihydroxybutyl nucleotide, 3,5-dihydroxypentyl nucleotide, 5xe2x80x2-5xe2x80x2-inverted nucleotide moiety, 5xe2x80x2-5xe2x80x2-inverted abasic moiety, 5xe2x80x2-phosphoramidate, 5xe2x80x2-phosphorothioate, 1,4-butanediol phosphate, 5xe2x80x2-amino; bridging and/or non-bridging 5xe2x80x2-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5xe2x80x2-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein). By the term xe2x80x9cnon-nucleotidexe2x80x9d is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. The terms xe2x80x9cabasicxe2x80x9d or xe2x80x9cabasic nucleotidexe2x80x9d as used herein encompass sugar moieties lacking a base or having other chemical groups in place of base at the 1xe2x80x2 position.
In connection with 2xe2x80x2-modified nucleotides as described for the present invention, by xe2x80x9caminoxe2x80x9d is meant 2xe2x80x2-NH2 or 2xe2x80x2-Oxe2x80x94NH2, which may be modified or un-modified. Such modified groups are described, for example, in Eckstein et al.; U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., WO 98/28317, respectively, wherein the U.S. Patent is incorporated by reference in its entirety.
The drawings will first briefly be described.